Sequence Conservation Analysis and Gene Relationships of Nucleocapsid (N) Gene in Orthocoronavirinae Subfamily

Abstract

Coronavirus (CoV) is a virus that causes respiratory and gastrointestinal diseases in animals and humans. It belongs to the Orthocoronavirina. The nucleocapsid protein (N) plays multiple roles in virus assembly, RNA transcription, and interaction with host cells. This study aimed to analyse the N protein by identifying conserved residues and exploring the gene and protein relationships within the Orthocoronavirinae. Therefore the results of this study are expected to help identify conserved regions of N protein in SARS-CoV-2 which can be used as probes for the virus identification process and can be used as target areas in vaccine development. We used 159 N gene and protein sequences, including 64 from Alpha, 51 from Beta-, 11 from Delta-, and 20 from Gammacoronavirus genera of the Orthocoronavirinae. Three sequences from Tobaniviridae were used as outgroups. Multiple sequence alignment (MSA) and phylogenetic tree analysis were performed using the neighbour-joining and Maximum Likelihood. The MSA results revealed several conserved residues, ranging from 18 to 41, were located in the N-terminal and Cterminal domains, the linker region, Nuclear Localization Signal (NLS), Nuclear Export Signal (NES) motifs, and Packing Signal (PS) binding sites. The phylogenetic tree analysis indicated that Gammacoronavirus and Deltacoronavirus were closely related to Betacoronavirus, while Alfacoronavirus showed the most distant relationship. Furthermore, the study identified 23 conserved residues involved in RNA binding, including amino acids such as Ser89, Val111, Pro112, Gly124, Tyr125, Phe150, Tyr151, Gly154, Thr155, Gly156, Trp180, Val181, Gly409, Arg411, Asn419, Gly421, and Pro443. These residues interacted with phosphate groups, nitrogenous bases, and pentose sugars and exhibited non-specific interactions with RNA. In summary, this study investigated the N protein in the Orthocoronavirinae subfamily, providing insights into its function, structure, and evolutionary relationships.